Nitric oxide (NO) is a simple diatomic molecule that is a powerful signaling compound and cytostatic agent found in nearly every tissue including endothelial cells, neural cells and macrophages. Mammalian cells synthesize NO using a two step enzymatic process that oxidizes L-arginine to N-ω-hydroxy-L-arginine, which is then converted into L-citrulline and an uncharged NO free radical. Three different nitric oxide synthase enzymes regulate NO production. Neuronal nitric oxide synthase (NOS1, or nNOS) is formed within neuronal tissue and plays an essential role in neurotransmission; endothelial nitric oxide synthase (NOS3 or eNOS), is secreted by endothelial cells and induces vasodilatation; inducible nitric oxide synthase (NOS2 or iNOS) is principally found in macrophages, hepatocytes and chondrocytes and is associated with immune cytotoxicity.
Neuronal NOS and eNOS are constitutive enzymes that regulate the rapid, short-term release of small amounts of NO. In these minute amounts NO activates guanylate cyclase which elevates cyclic guanosine monophosphate (cGMP) concentrations which in turn increase intracellular Ca+2 levels. Increased intracellular Ca+2 concentrations result in smooth muscle relaxation which accounts for NO's vasodilating effects. Inducible NOS is responsible for the sustained release of larger amounts of NO and is activated by extracellular factors including endotoxins and cytokines. These higher NO levels play a key role in cellular immunity.
Nitric oxide's therapeutic potential has been studied in a diverse number of clinical indications including cancer, coronary artery heart disease, restenosis, hypertension, angiogenesis, and sexual dysfunction. Moreover, recent studies have demonstrated that NO also possesses considerable in vivo and ex vivo antimicrobial activity (Fang, F. C., 1997. Perspectives series: host/pathogen interactions. Mechanisms of Nitric Oxide-antimicrobial activity. J Clin Invest Jun. 15; 99 (12):2818-25; Fang, F. C., 1999. Nitric Oxide and Infection, Kluwer Academic/Plenum Publishers: New York; see also: U.S. Pat. No. 5,814,666 [the “'666 patent”] filed Apr. 24, 1995 and issued to Green at al.; the entire contents of both are hereby incorporated by reference) and is thus suitable for use in the treatment of infectious diseases.
Nitric oxide's unique combination of physiological properties has made it an ideal candidate for treating vascular diseases, specifically ischemic heart disease. Ischemic heart disease results when blood flow to the heart is restricted, usually as a result of a blockage in the one or more coronary arteries. Most forms of ischemic heart disease are treated using coronary artery bypass graft (CABG) surgery or by restoring blocked vessel patency using transluminal coronary angioplasty (PTCA) and/or stent placement.
However, CABG and PTCA can fail due to restenosis, a multi-factorial process whereby the previously opened vessel lumen narrows and becomes re-occluded. Restenosis has been found to occur in approximately 30% to 50% of angioplasty and other transcatheter patients within three to six months (Currier, J. W. et al. 1995. Restenosis after percutaneous transluminal coronary angioplasty: have we been aiming at the wrong target? J Am Coll Cardiol; 25:516-520). Restenosis is initiated when thrombocytes (platelets) adhere to a vessel injury site caused by balloon inflation and initiate thrombogenesis (clot formation) and/or vascular smooth muscle cell over-proliferation (hyperplasia). As a result, the previously opened lumen begins to narrow, restricting or occluding the injured vessel. Recently, researchers have demonstrated that the anti-thromobogenic and anti-smooth muscle proliferative effects of NO can significantly reduce restenosis in animals (Bohl, K. S. et al. 2000. Nitric oxide-generating polymers reduce platelet adhesion and smooth muscle cell proliferation. Biomaterials; 21(22): 2273-8; Buergler, J. M. et al. Use of nitric-oxide-eluting polymer-coated stents for prevention of restenosis in pigs. Coron. Artery Dis.; 11(4): 351-7; Janero, D. R. et al. Nitric oxide and postangioplasty restenosis: pathological correlates and therapeutic potential. 29(12): 1199-221; Le Tourneau, T. et al. J. Am. Coll. Cardiol. Role of nitric oxide in restenosis after experimental balloon angioplasty in the hypercholesterolemic rabbit: effects on neointimal hyperplasia and vascular remodeling. 33 (3): 876-82). Consequently, significant interest has been directed at developing medical devices that focus the anti-restenotic effects of NO directly on anatomical sites at greatest risk for restenosis.
Another method for treating an ischemic organ, especially the heart, is to revascularize the affected area by inducing the growth of new blood vessel and capillaries. This process, called angiogenesis, has received considerable attention as an alternative to CABG and PTCA for the treatment of ischemic heart disease (Dulak, J. et al., 2000. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol; March;20(3): 659-666). Compounds shown to induce or up-regulate angiogenesis include nitric oxide, fibroblast growth factor (FGF) vascular endothelial growth factor (VEGF) and members of the epidermal growth factor (EGF) family such as transforming growth factor alpha (TGF alpha), transforming growth factor beta (TGF beta), betacellulin, amphiregulin, and vaccinia growth factor among other factors. Nitric oxide is involved in the regulation of these biochemicals; therefore under certain conditions NO may exert angiogenic effects.
The in vivo use of NO-releasing compounds to induce angiogenesis, prevent restenosis, reduce unwanted thrombogenicity, and promote wound healing appears to be promising. However, the current NO-releasing techniques have failed to fully mimic the natural NO release associated with endothelial cells. As previously mentioned, natural NO production is regulated by a combination of constitutive and inducible enzymes. Endothelial cells, the primary regulator of vascular homeostasis, are provided with continuous low levels of NO release through constitutive enzyme (eNOS) activity. Moreover, when required, inducible enzymes (iNOS) can provide cells with momentary bursts of NO. In principle, such transient physiological bursts of NO can be mimicked using polymers or metallic surfaces coated with NO-donating materials that release NO immediately upon exposure to aqueous physiological environments. However, the sustained delivery of low levels of NO by endothelial cells has proven to be much more difficult to simulate with NO-releasing materials.
Another daunting task has proven to be the preferential or selective delivery of NO to specific target organs. Nitric oxide reacts readily with a variety of biomolecules and can be toxic when administered systemically. Previous efforts to provide therapeutic NO levels have generally relied on NO prodrugs such as glyceryl trinitrate and sodium nitroprusside. These compounds, unlike NO gas, are generally stable; however, their pharmacological activity is usually short lived. Moreover, the enzymes and co-factors necessary to convert glyceryl trinitrate into NO are rapidly depleted. Hence, repeated use of this compound over short time periods results in the development of drug tolerance. Prolonged use of sodium nitroprusside can lead to the excessive generation of highly toxic cyanide which can accumulate as a result thus limiting its long-term use. Consequently, significant attention has been directed towards the development of NO generators or donor compounds that can be used for the sustained localized therapeutic administration of NO without toxic side effects.
Nitric oxide-releasing compounds suitable for in vivo applications have been developed by a number of investigators. As early as 1960 it was demonstrated that nitric oxide gas could be reacted with amines to form NO-releasing anions having the following general formula:R—R′N—N(O)NO−  Formula 1Salts of these compounds spontaneously decompose and release NO in solution. (R. S. Drago et al J. Am. Chem. Soc. 1960, 82, 96-98.)
Nitric oxide-releasing compounds with sufficient stability in aqueous physiological buffers to be useful as therapeutics were ultimately developed by Keefer et al. as described in U.S. Pat. Nos. 4,954,526, 5,039,705, 5,155,137, 5,212,204, 5,250,550, 5,366,997, 5,405,919, 5,525,357 and 5,650,447 and in J. A. Hrabie et al, J. Org. Chem. 1993, 58, 1472-1476, all of which are herein incorporated by reference. Briefly, Hrabie et al. describes NO-releasing intramolecular salts (zwitterions) having the general formula:R N[N(O)NO]−(CH2)xNH2+R′  Formula 2Stable NO-releasing compounds of Formula 2 (nitric oxide/nucleophile complexes) have been coupled to a wide range of amine containing polymers whose backbone molecular structure is non- or poorly cross-linked (Smith D. J. et al. 1996 Nitric oxide-releasing polymers containing the [N(O)NO]− group. J Med Chem 39:1148-1156, Pulfer, S. K. et al. 1997 Incorporation of nitric oxide-releasing crosslinked polyethyleneimine microspheres into vascular graft. J. Biomed Mater Res. 37:182-9, Mowery, K. A. et al. 2000. Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release. Biomaterials 21:9-21, Bauer, J. A. et al. 1998. Evaluation of linear polyethyleneimine/nitric oxide adduct on wound repair:therapy verses toxicity. Wound Repair and Regeneration, Vol. 6 No. 6:569-576. The[N(O)NO]− (abbreviated herein after at NONO) containing compounds thus described release NO via a first order reaction that is predictable and easily quantified. Such NO-donor compounds are commonly known in the art as diazeniumdiolates.
U.S. Pat. No. 5,405,919 (“the '919 patent”) is an example of a patent that describes methods for bonding or coupling diazeniumdiolate NO-releasing groups to biologically acceptable polymers. Examples of such polymers include polyolefins, such as polystyrene, polypropylene, polyethylene, polyterafluoroethylene, polyvinylidene, or derivatized polyolefins such as polyethyleneimine, polyesters, polyethers, polyurethanes and the like. The NO-release totals for the polymers cited in the '919 patent were measured in the range of 3 to 11 nmol/mg. Implantable medical devices composed of biologically acceptable forms of such polymers represent a potential means for the site-specific NO delivery to a particular tissue or target organ.
Significant progress has been made in the preparation, formulation, and protective group derivatization of diazeniumdiolated amines. U.S. Pat. No. 5,155,137 discloses general methods of preparing polyamine/nitric oxide complexes suitable for treating cardiovascular diseases. The '666 patent describes a novel means of formulating a diazeniumdiolated amine by impregnating such moieties in multilamellar liposomes. Liposome encapsulated diazeniumdiolates are shielded from the aqueous milieu until phagocytized by a macrophage. Once inside the macrophage, the liposome encapsulated diazeniumdiolate releases its contents into the lumen of the phagolysome and there is a corresponding generation of NO as the “free” diazeniumdiolate moieties. In vitro studies have shown that the method described in the '666 patent is a particularly effective means of killing phagocytized pathogenic microorganisms within macrophages. Liposome encapsulation of diazeniumdiolates appears to function entirely as a means of enhancing the delivery of such compounds to cells as there is no corresponding extension in the half-life of NO release vs. non-encapsulated diazeniumdiolates.
U.S. Pat. No. 5,366,997 discloses techniques by which the distal oxygen atom of the diazeniumdiolate anion can be derivatized through the covalent attachment of a protective group. Such modified diazeniumdiolates are generally quite stable when exposed to aqueous buffers under conditions of physiological temperature and pH. However, certain organs are able to metabolically remove the protective group from protected diazeniumdiolates with the concomitant formation of the parent diazeniumdiolate moiety.
The availability of stable NO-releasing diazeniumdiolates has greatly advanced the potential for developing NO-delivering medicaments. However, there is still a need for even better control over the duration of in vivo NO release. Specifically, there is a need to develop NO-donating polymeric materials that are capable of sustained NO release in physiological buffer solutions for periods lasting several months to years. Moreover, there is a need to develop materials that can better mimic in situ NO production where NO bursts are followed by sustained lower level release of NO delivered directly to a target organ or group of cells.